This is the second project in a Centers of Cancer Nanotechnology Excellence (CCNE) application entitled Emory-GA Tech Nanotechnology Center for Personalized and Predictive Oncology." The primary objective of this project is to develop activatable nanoprobes for cancer detection and analysis, including dual FRET molecular beacons, peptide-linked molecular beacons, NIR-dye labeled molecular beacons and quantum-dot / lanthanide-chelate nanoprobes. Molecular beacons are dual-labeled oligonucleotide probes with a stemloop hairpin structure. Hybridization of molecular beacons with target mRNAs corresponding to cancer genes results in fluorescence of the cell. Thus, cancer cells (bright) can be distinguished from normal cells (dark). However, the conventional design of molecular beacon may induce a significant amount of false positives in cancer cell detection due to probe degradation by nucleases and non-specific interactions. To overcome this difficulty, we have developed a dual-FRET beacon technology in which a pair of molecular beacons with respective donor and acceptor fluorophores hybridizes to adjacent regions of the same target mRNA, and results in a FRET signal upon proper excitation, which is differentiable from none-FRET false-positive signals. We have also developed new molecular beacon delivery methods with high efficiency and fast kinetics for live-cell studies, and have examined the sensitivity and specificity of detecting K-ras and survivin mRNAs in living cells. In this CCNE project, we will demonstrate the quantitative capability of molecular beacons in detecting and analyzing multiple cancer genes in living cells, and will establish the ability of molecular beacons in detecting mutant mRNAs in fixed or live cells, as well as in in-vivo cancer detection,. We will further develop near-inferred (NIR) beacons, demonstrate tissue delivery capabilities, determine the signal-to-background ratio, and examine the possible cytotoxicity, antisense and RNA interference (RNAi) effects of these beacons in living cells. To further increase the detection sensitivity, we will develop new activatable nanoprobes using quantum dots and lanthanide chelates, so that low abundance genes can be quantified in living cells. The goal is to develop activatable nanoprobes to detect tumor-marker genes in cells, tissue specimens and animals with high specificity and sensitivity, thus generating a better understanding of tumor biology, and leading to better cancer diagnosis and therapy.